Nucleolar Localization Of PNRC And Its Role In The Nucleolus & Identification And Characterizaton Of PNRC Splicing Variants

Nuclear receptors (NRs) comprise a large class of transcription factors that play central roles in development, reproduction and homeostasis. NR dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. Ligand-dependent dynamic exchange of corepressors for coactivators serves as a well-regulated switch from gene repression to gene activation.PNRC (proline-rich nuclear receptor coregulatory protein), a 35.2 kDa proline-rich protein, was primarily identified as a coactivator of nuclear receptors (NRs), which could interact in a ligand-dependent manner with the ligand-binding domain of NRs including ERα, ERβ, PR, GR, TR, RARα, RARβ, RXR, RORα, RORβ, HNF4α, HNF4γand LRH1, and in a ligand-independent manner with the orphan receptors SF1, ERRα1 and ERRγ. In addition to enhancing NR-meditated RNA polymerase II transcription, PNRC has been found to be capable of down-regulating the activation of Ras and MAP kinase through interacting with Grb2, an important adapter protein involved in growth factor/Ras signaling pathway. Recently PNRC has also been demonstrated to stimulate RNA polymerase III-dependent transcription through interacting with the subunit RPC39 of RNA polymerase III.PNRC exerts its functions mainly in the nucleus, and previous studies also showed that it was predominantly localized in the nucleus. Theoretically, small proteins (< 40 kDa) could go through nuclear pore complexes by passive diffusion. Therefore, it is reasonable to speculate that there is a signal to retain 35.2 kDa PNRC in the nucleus. In the current work, during identifying the signal controlling the nuclear localization of PNRC, we find that PNRC localizes in the nucleolus and also involves pre-rRNA synthesis by RNA polymerase I. Through a series of mutation analysis, we identify the sequence at position 94-101 (94PKKRRKKK101) as its nucleolar targeting sequence (NTS) as well as nuclear localization sequence (NLS). Fusion of this NTS/NLS to GFP could direct GFP to the nucleus and nucleolus. Alanine scanning mutagenesis of the NLS/NTS sequence reveals that lysine-100 is the key residue of PNRC for nucleolar localization, but not for nuclear localization. Moreover, we demonstrate that the stretches of six successive basic residues like KKRRKK, KRRKKK, KKKKKK, and RRRRRR are sufficient for targetting GFP to the nucleolus, which could therefore function as a NTS. Through immunoprecipitation, we find that PNRC is associated with B23/nucleophosmin, a major phosphoprotein in the nucleolus, through its NTS. Inhibition of rDNA transcription by actinomycin D causes translocation of GFP-tagged PNRC from nucleolus to nucleoplasm, whereas proteasome inhibitor MG132 treatment promotes GFP-PNRC to accumulate in the nucleolus, indicating that nucleolar localization of PNRC is dependent on active rRNA transcription and proteotoxic stress affects nucleolar localization of PNRC. In addition, we find that knockdown of PNRC expression by siRNA dramatically reduces pre-rRNA synthesis in breast cancer cell line MCF-7 cells. Concomitantly C23/nucleolin, a nucleolar protein involved in rRNA synthesis, is also decreased after knockdown of PNRC. Moreover, we demonstrate that PNRC overexpression leads to increased C23 protein level. Our results suggest that PNRC is localized to the nucleolus via its NTS that binds B23/nucleophosmin, and is involved in rRNA synthesis by regulating C23/nucleolin protein level.In the current work, we also investigate alternative splicing of PNRC. On the base of bioinformatics, we identify three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f using the strategy of cloning plus sequencing. PNRC1c and PNRC1f are generated through alternative recognition of the 3'-splice site in exon 1, leading to in-frame deletion of 79 amino acids (aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of PNRC. We find that these isoforms differ in their abilities to bind NRs and potentiate NR-mediated transcriptions. Moreover, we find that PNRC1d can modulate the activity of full-length PNRC in enhancing ER-mediated transcription. The results of this part suggest PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity.